Selection of a preparative column

Selecting the separation mode of chromatography
(investigation of separation conditions using various analytical columns)
When more than one mode is available for the separation of samples, the following key factors should be considered for the selection of an appropriate separation mode

1. Resolution: Selectivity of the packing material for the compound of interest
2. Load: Capacity of the packing material
3. Speed: Isolation time

Column size
The Table below provides a rough guide for selection of column inner diameters and packing material particle sizes.

1. Column inner diameter Sample load is proportional to the column cross-sectional area. It is necessary to select a column inner diameter suitable for the sample load.
   
   
2. Packing material particle size Smaller particle sizes can provide higher column efficiency, but provided at higher prices. More over, due to higher pressures applied to columns with smaller particle sizes, equipment needs to be resistant to the pressure. When the distance between target component and the nearest peaks are very close, that requires higher resolution, packing materials with small particle size are effective. In contrast, column efficiency lowers with larger particle sizes, but provided at lower prices with lower column pressure.
   
   
3. Column length Better separability can be obtained with longer column length and higher sample load can be tolerable, but the longer the column, the higher the column pressure becomes.

Optimization of separation conditions

Investigation of preparative separation conditions with packing material with the selected size. Optimize preparative separation conditions with analytical sized columns. R&D series columns are recommended in order to obtain more efficient results.

1. Optimization of elution condition Unlike analysis, the top priority of preparative separation is to obtain target component efficiently at a high purity and recovery . A key to perform an efficient preparative separation is to set the elution conditions in consideration of which components should and should not be separated. Packing material with larger particle size compared to analytical purposes compromise the column efficiency to lose required separation. In such case, improve the separation by adjusting elution conditions and/or reduce linear flow rate. If the separation is not improved then, change the column length and/or particle size.
2. Investigate the load Increase the load stepwise and work out the maximum load to obtain the desired purity. Calculate the load based on the maximum load worked out in case of scaling up to a preparative column.
3. Inspection on preparative system Carry out an inspection to verify that the flow rate and the column pressure to be applied at the actual preparative separation are compliance to the system performance. In order to obtain the same separation as the column for investigation, the linear flow rate of preparative column should be same as the column for investigation. Same linear flow rate is obtained by setting the flow rate of preparative column as much as the cross-sectional ratio of the flow rate of the column for investigation. If the linear flow rate is same, the pressure of the preparative column will be the same as the column for investigation. If the column pressure exceeds the pressure capacity of the system, the flow rate, column length, etc. need to be changed. Piping system should be changed to comply with the applied flow rate.

Perform preparative separation

Carry out the preparative separation and evaluate the yield and purity.